We have discovered novel pure strains of A. pullulans which produce decreased amounts of pigment. We have further discovered a method for selecting the inventive pure strains; a method for making pullulan which is sufficiently non-pigmented so as to require little or no decolorization for use; and a method for making such pullulan having an average molecular weight in excess of about 8xc3x97105, as well as the pullulan made by the inventive processes.
More particularly, we have discovered that the pigment production characteristic of A. pullulans is associated mainly with the filamentous form and chlamydospores. We have further discovered that wild-type strains of A. pullulans obtained, for example, from depositories or natural sources, can be treated to enrich the fraction of yeast-like cells therein, and the yeast-like cells can be separated from the non-yeast-like cells. The separated yeast-like cells may then be grown and colonies can then be visually isolated based on their degree of pigmentation. The isolated colonies may then be subjected to further purification, if desired, and tested to measure and further isolate the strain producing the least amount of pigment when subjected to fermentation conditions. It is also desirable to measure the pullulan yields of the isolated colonies so that the strain having the maximum product yield as well as the minimum pigment production can be determined.
In accordance with the invention, pullulan of very high molecular weight may be obtained by subjecting A. pullulans to fermentation conditions and neutralizing the fermentation broth to a pH value of about 7 after the fermentation broth has stabilized at its characteristic low pH.
In yet another embodiment of the production method, pullulan of very high molecular weight may be obtained by subjecting A. pullulans to fermentation conditions and heat treating the accumulated pullulan for a time period and at a temperature sufficient to deactivate any pullulan decomposing enzymes in the accumulated pullulan.
By these methods, a novel pullulan product having an average molecular weight in excess of about 4xc3x97106 may be obtained and this can be accomplished without adversely affecting the yield. When the inventive pure strain is used as the production culture, a pullulan product having both high molecular weight and minimal pigmentation is obtained. The inventive pullulan exhibits a high viscosity and is particularly suitable for use in films, fibers, and as a rheology control agent for a variety of materials.